Transcriptional regulation of development and homeostasis in complex eukaryotes, including humans and other mammals, birds, fish, insects, and the like, is controlled by a wide variety of regulatory substances, including steroid and thyroid hormones. These hormones exert potent effects on development and differentiation of phylogenetically diverse organisms. The effects of hormones are mediated by interaction with specific, high affinity binding proteins referred to as receptors.
The ability to identify additional compounds which are able to affect transcription of genes which are responsive to steroid hormones or metabolites thereof, would be of significant value in identifying compounds of potential therapeutic use. Further, systems useful for monitoring solutions, body fluids, and the like, for the presence of steroid hormones or metabolites thereof, would be of value in medical diagnosis, as well as for various biochemical applications.
A number of receptor proteins, each specific for one of several classes of cognate steroid hormones [e.g., estrogens (estrogen receptor), progesterones (progesterone receptor), glucocorticoid (glucocorticoid receptor), androgens (androgen receptor), aldosterones (mineralocorticoid receptor), vitamin D (vitamin D receptor)], retinoids (e.g., retinoic acid receptor) or for cognate thyroid hormones (e.g., thyroid hormone receptor), are known. Receptor proteins have been found to be distributed throughout the cell population of complex eukaryotes in a tissue specific fashion.
Molecular cloning studies have made it possible to demonstrate that receptors for steroid, retinoid and thyroid hormones are all structurally related and comprise a superfamily of regulatory proteins. These regulatory proteins are capable of modulating specific gene expression in response to hormone stimulation by binding directly to cis-acting elements. Structural comparisons and functional studies with mutant receptors have revealed that these molecules are composed of a series of discrete functional domains, most notably, a DNA-binding domain that is composed typically of 66-68 amino acids, including two zinc fingers and an associated carboxy terminal stretch of approximately 250 amino acids, which latter region comprises the ligand-binding domain.
An important advance in the characterization of this superfamily of regulatory proteins has been the delineation of a growing list of gene products which possess the structural features of hormone receptors. This growing list of gene products has been isolated by low-stringency hybridization techniques employing DNA sequences encoding previously identified hormone receptor proteins.
It is known that steroid or thyroid hormones, protected forms thereof, or metabolites thereof, enter cells and bind to the corresponding specific receptor protein, initiating an allosteric alteration of the protein. As a result of this alteration, the complex of receptor and hormone (or metabolite thereof) is capable of binding to certain specific sites on chromatin with high affinity.
It is also known that many of the primary effects of steroid and thyroid hormones involve increased transcription of a subset of genes in specific cell types.
A number of steroid hormone- and thyroid hormone-responsive transcriptional control units have been identified. These include the mouse mammary tumor virus 5'-long terminal repeat (MTV LTR), responsive to glucocorticoid, aldosterone and androgen hormones; the transcriptional control units for mammalian growth hormone genes, responsive to glucocorticoids, estrogens and thyroid hormones; the transcriptional control units for mammalian prolactin genes and progesterone receptor genes, responsive to estrogens; the transcriptional control units for avian ovalbumin genes, responsive to progesterones; mammalian metallothionein gene transcriptional control units, responsive to glucocorticoids; and mammalian hepatic .alpha..sub.2u -gloubulin gene transcriptional control units, responsive to androgens, estrogens, thyroid hormones, and glucocorticoids.
A major obstacle to further understanding and more widespread use of the various members of the steroid/thyroid superfamily of hormone receptors has been a lack of availability of the receptor proteins, in sufficient quantity and sufficiently pure form, to allow them to be adequately characterized. The same is true for the DNA gene segments which encode them. Lack of availability of these DNA segments has prevented in vitro manipulation and in vivo expression of the receptor-encoding genes, and consequently the knowledge such manipulation and expression would yield.
In addition, a further obstacle to a more complete understanding and more widespread use of members of the steroid/thyroid receptor superfamily is the fact that additional members of this superfamily remain to be discovered, isolated and characterized.
The present invention is directed to overcoming these problems of short supply of adequately purified receptor material, lack of DNA segments which encode such receptors and increasing the number of identified and characterized hormone receptors which are available for use.